Cytochrome c oxidase (cytochrome aa3; EC 1.9.3.1) is a terminal oxidase enzyme in the aerobic respiratory electron transport system of mitochondria and many bacteria. The enzyme is a cytoplasmic membrane spanning complex that catalyzes the final step in electron excretion involving the re-oxidation of ferrocytochrome c (electron donor) at the periplasmic surface and the reduction of molecular oxygen (electron acceptor) to water at the cytoplasmic surface. The reaction is coupled to the extrusion of protons across the membrane. This coupling is indispensable for the conservation of biological energy derived from substrate oxidation.
Various types of cytochrome complex, e.g. aa3, a1, caa3, o, bo, co, and bd-types, have been identified as functional terminal oxidases. The purification and characterization of some terminal oxidases has been reported. Matsushita et al. reported that Acetobacter aceti IFO 3283 contains two terminal oxidases, cytochrome a1 and o. (Proc. Natl. Acad. Sci., USA, 87: 9863, 1990; J. Bacteriol. 174: 122, 1992). (Id.) Matsushita et al. purified and characterized the cytochrome a1. Matsushita et al. also reported the purification of cytochrome o from Gluconobacter (Biochem. Biophys. Acta, 894: 304, 1987). Tayama et al. disclosed the terminal oxidase (cytochrome a1) genes of A. aceti (JP 93-317054) and they also purified the oxidase enzyme consisting of four subunits of 72, 34, 21, and 13 kDa and also containing heme a and heme b. The oxidases in Acetobacter and Gluconobacter belong to quinol oxidase family of oxidases. Cytochrome aa3 (cytochrome c oxidase) has been purified from bovine heart, yeast, and many bacteria including Paracoccous denitrificans (Solioz et al., J. Biol. Chem., 257: 1579-1582, 1982) and Rhodobacter sphaeroides (Hosler et al., J. Biol. Chem., 267: 24264-24272, 1992).
Mammalian (mitochondrial) cytochrome c oxidase (aa3-type) complex contains 13 different subunits; the three core subunits I, II and III (CO I, II and III) are encoded by mitochondria DNA, while the remaining 10 subunits originate from the nucleus. Bacterial aa3-type cytochrome c oxidase also contains three core subunits that are homologous to the mitochondrial core subunits. However, it is reported that CO III was easily lost during purification, resulting in preparations composed of CO I and CO II only (Ludwig et al., Proc. Natl. Acad. Sci. USA, 77: 196-200, 1980). The cytochrome c oxidase complex consisting of the two-subunit (CO I and II) showed redox activity along with the generation of an electrochemical proton gradient. In the case of P. denitrificans (Haltia et al., The EMBO Journal, 10: 2015-2021, 1991) and R. sphaeroides (Cao et al., Gene, 101: 133-137, 1991), both two-subunit-type (CO I/II) and three-subunit-type (CO I/II/III) complexes were isolated by different purification methods. Genetically, genes for CO II and III are located in an operon, while the gene for CO I is independently located (Raitio et al., The EMBO Journal, 9: 2825-2833, 1987; Shapleigh et al., Proc. Natl. Acad. Sci. USA, 89: 4786-4790, 1992).
Terminal oxidases, as described above play an important role in cellular growth under aerobic conditions by accomplishing the reduction of the molecular oxygen. In oxidative fermentation, the respiratory chain, including the terminal oxidase, function by completing oxidation of a substrate to produce an oxidized product. In this context, it is very important to improve the efficiency of the respiratory chain in order to achieve efficient oxidative fermentation.
G. oxydans DSM 4025 produces 2-keto-L-gulonic acid (hereinafter: 2KGA), an important intermediate in the process of L-ascorbic acid production from L-sorbose via L-sorbosone (T. Hoshino et al., EP 0 366 922 A). The oxidation of the substrate, L-sorbose, to 2KGA was thought to be accomplished by the respiratory electron transport chain. The terminal oxidase that catalyzes the final electron excretion step via oxygen, might be one of the kinetic rate-limiting steps in the 2KGA production system as well as in the production of other redox components. The primary dehydrogenase responsible for 2KGA formation from L-sorbose was isolated (T. Hoshino et al., EP 606621 A) and the genes were cloned and sequenced. Four isozymes of the primary dehydrogenase were found (T. Hoshino et al., EP 832974 A). Their direct electron acceptor, cytochrome c551, was also purified and its gene cloned (T. Hoshino et al., EP 0869175 A). However, the terminal oxidase was not isolated and its genes were not cloned.